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Title: Evading drug efflux with drug-binding peptides
Author: Fairbrother, Sally M.
ISNI:       0000 0004 2683 3465
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2003
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The efflux of drugs, particularly multi-drug resistant (MDR) drugs, by transporters such as P-glycoprotein provides an additional hurdle to achieving high levels of drug bioavailability. The aim of this project was to investigate drug-binding peptides as potential delivery tools, which might circumvent drug efflux by transporters such as Pglycoprotein. Popkov et 01 1998 reported a number of phage-displayed peptides (I Omers), which bound to doxorubicin. A literature search was carried out and several other potential drug-binding sequences were identified. Peptides were synthesised and then screened for drug-binding and their effect on drug interactions with P-glycoprotein. Due to the small and hydrophobic nature of the drug and peptide molecules, both homogeneous and heterogeneous drug-peptide binding assays were investigated. Drugpeptide binding was identified in one sequence using circular dichroism, size exclusion HPLC, equilibrium dialysis and a new biosensor, the Farfield AnalightBi0200. Competition assays using the fluorophores rhodamine 123 and calcein-AM suggested that MDR drugs formed complexes with these fluorophores. Drug-peptide aggregates were also identified by particle sizing, using photon correlation spectroscopy. Drug-P-glycoprotein interactions were investigated using a cell-based competition assays, cytotoxicity and MDCK cell monolayer transport studies. All the potential drug-binding peptide sequences were screened using a calcein-AM competition assay in PGP over-expressing cells (CHO CHRC5), whereby calcein-AM and drug (± peptide) compete to interact with P-glycoprotein. Only the Popkov-reported sequences were found to reduce drug-P-glycoprotein interactions. However, these sequences did not enhance the cytotoxicity of doxorubicin or vinblastine (cytotoxic P-glycoprotein substrates) in Caco-2 or CHO CHRC5 cells. MDCK monolayer studies found the peptide to have a slight but not significant effect on 3H vinblastine basal to apical transport. This difference may be due to greater sensitivity of the calcein AM compared to the monolayer transport assay. With a view to enhancing the delivery properties of the drug-binding peptides, new sequences containing cell-penetrating motifs were synthesised. The Penetratin sequence was synthesised in tandem to a drug-binding peptide sequence, whilst the Simian virus 40 motif was added to the peptide using a linker. Both these peptides gave similar results in the calcein-AM competition assay as the drug-binding peptide alone. The ability of the peptides to penetrate cell membranes was tested using a calcein-liposome leakage assay. Both the new sequences were slightly lytic, but complete liposome lysis particularly resulted when tamoxifen was added. Interestingly, a similar result was achieved with the drug-binding peptide sequence alone. Some drug-binding peptides appear to affect the behaviour of P-glycoprotein in suspended-cell transport models, when assayed by calcein-AM. However, the interaction between drugs and peptides in other cell-based models appears more complex.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available